Regulatory T cells (Tregs) function by suppressing potentially detrimental actions of T helper cells. In this study, we investigated the ability of two naturally occurring compounds, Spartiene Sulfate and Harpagoside, to induce Tregs from human peripheral blood mononuclear cells (PBMCs) in vitro. To achieve this, PBMCs were isolated from healthy donors and cultured for five days with each of the two compounds in the presence or absence of the T cell mitogen Concanavalin A. Induction of Tregs was then evaluated by measuring the expression of Treg cell markers and cytokines using RT-PCR, Flow Cytometry and ELISA. Suppressive function of induced Tregs was then investigated by unidirectional mixed lymphocyte reaction (MLR). RT-PCR analysis revealed upregulation of key T regulatory cell markers including Forkhead box P3(FoxP3), Transforming Growth Factor-Beta (TGF-ß) and Interleukin-10 (IL-10). Flow Cytometric analysis revealed an increase in the percentage of cells expressing a CD4+ CD25hi FoxP3+ CD127- Treg cell profile. ELISA analysis showed elevated production of IL-10 cytokine in ConA activated PBMCs when treated with SS or Harp. Furthermore, one-way MLR revealed significant suppression of immune cell proliferation. Our results indicate that both SS and Harp can induce Tregs in vitro that possess functionally suppressive ability.